FERTILITY & REPRODUCTION Oocyte Vitrification: A Comparative Analysis Between Fresh and Cryopreserved Oocytes in an Oocyte Donation Program Deepa Talreja 1, Chirag Gupta 2, *, Hrishikesh Pai 1, Nandita Palshetkar 1 1 Department of IVF, Lilavati Hospital, Mumbai, India 2 Department of Urology and Andrology, Lilavati Hospital, Mumbai, India ABSTRACT Background: Oocyte Cryopreservation has become an important part of infertility treatment for various reasons such as fertility preservation in women going for oncological treatment; in oocyte donation cycles; in eliminating several religious, ethical, and legal concerns of embryo freezing and in women who wish to delay childbirth. The newer vitrification technique for freezing has further improved the success rates for actual conception than the earlier method of slow freezing. A successful oocyte freezing program can help in establishment of oocyte banks, which would help to provide compatible oocytes immediately, thus would eliminate the several problems of fresh donor cycles. Methods: In this retrospective observational study, total 60 oocyte donation cycles were included (38 were fresh and 22 were vitrified oocytes cycle, respectively). After a thorough screening, controlled ovarian hyperstimulation for donors was performed using flexible antagonist protocol. All mature oocytes were allocated into vitrified oocytes and fresh oocytes groups. Vitrification technique using Cryotop method was used for oocyte freezing. Both clinical and laboratory outcomes of vitrified and fresh oocytes in donor cycles were compared. Results: A total of 600 oocytes (226 vitrified oocytes and 374 fresh oocytes), were studied. After warming 218 oocytes survived resulting in survival rate of 96.4%. Fertilization rate and embryo formation rate was 86.2% and 93.6%, respectively. Results of frozen-thawed oocyte donor cycles were compared with fresh donation cycles. For fresh oocyte group, fertilization rate and embryo formation rate was 83.4% and 92.6%, respectively. On comparing clinical outcomes, clinical pregnancy rate was 60.5% in fresh group and 63.6% in vitrified group. Conclusion: Both clinical and laboratory results obtained in the study suggest that oocyte cryopreservation can be performed with reproducible success, thus vitrification technique can be provided as a useful tool for achieving highly successful outcomes in an oocyte donor program. Keywords: Oocyte Cryopreservation; Vitrification; Fertility; Preservation. प ष ठभ म स र (ABSTRACT IN HINDI) म स त त क इल ज ऊस इट (अ ड ) फ र ज ग कई मततम मतय एक हत वप र ण प रक र य ह, ज स क रक क रकस भ प रक र क क न सर स ग रमसत महल ओ क मलए फ र टटमलट प र व श क र प, ड र आईव एफ प रक र य, य क र र कई मतक व ध र म क क रर स जह भ र र क फ र ज ग क सह ह ज त, ऐस मत मत ऊस इट फ र ज ग एक हत वप र ण मवकल प ह ज महल ए अप ब क म र णय क द र स प र कर च हत ह, वह भ इस मवकल प क चय कर सकत ह व मवटर क रफ क श तक क ऊस इट (अ ड ) फ र ज ग क और अमधक स ल मवकल प ब य ह, क य क रक प र तल फ र ज ग तक क क त ल यह अमधक क रगर व स ल ह स ल ऊस इट फ र ज ग तक क, ऊस इट ब क क त प क आध र ब सकत ह, मजसस क रक ड र आईव एफ प रक र य आ व ल कई ब ध ओ क द र क रकय ज सकत ह This is an Open Access article published by World Scientific Publishing Company. It is distributed under the terms of the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 (CC BY-NC-ND) License which permits use, distribution and reproduction, provided that the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Received 10 June 2019; Accepted 14 February 2020; Published 12 March 2020 *Correspondence should be addressed to: Dr. Chirag Gupta, Department of Urology and Andrology, Lilavati Hospital in Mumbai, A-791, Bandra Reclamation, Bandra (W), Mumbai 40005, India. Email: drchirag845@gmail.com VOLUME 2 NUMBER 1 MARCH 2020 9 13 DOI: 10.1142/S2661318220500024
प रक र य यह श ध क ल ६० ऊस इट ड र प रक र य ओ पर क रकय गय ( मजस ३८ फ र श व २२ फ र ज प रक र य ए ह ) प रक र य श म ल सभ ड र महल ओ क प र ण ज च क ब द अ ड उत प क रदत कर क मलए अ ट ग म तट प र ट क ल क उपय ग क रकय गय सभ प र णत: मवकमसत( च य र) अ ड क द श र मर य मवभ मजत क रकय गय अ ड क फ र जजग क मलए क र य ट प तक क क उपय ग क रकय गय पररर क ल ६०० अ ड क अध यय क रकय गय मजस २२६ फ र ज ऊस इट व ३७४ फ र श ऊस इट व र म ग क प रक र य क ब द २१८ अ ड ज मवत रह स ल रह अ णत ज व दर ९६.४ % रह फ र टटल य श व भ र र ब क दर श ८६.२% व ९३.६ % रह फ र ज व फ र श ऊस इट क प रक र य ओ क आ कड क आपस त ल क गय फ र श ऊस इट क मलए म ष च (फ र टटल य श ) दर व भ र र ब क दर श ८३.४ % व ९२.६% रह फ र श व फ र ज प रक र य गभ णवत दर ( प र ग नन स दर) श ६०.५ % व ६३.६ % रह म ष कषण इस श ध फ र ज व फ र श ऊस इट ड र प र ग र क आकड क त ल कर पर यह म ष कषण म कलत ह क रक, ऊस इट फ र ज ग एक स ल व क रगर तक क ह इसमलए इस तक क क ऊस इट ड र प रक र य एक हत वप र ण मवकल प क र प उपय ग क रकय ज सकत ह म ख य शब द व टर व क शन; ऊस इट (अ ड ) फ र व ग; वट व ट प र शन INTRODUCTION Assisted reproductive technique (ART) continues to attract immense attention scientifically and socially (David et al., 2012). Many breakthroughs have come in the form of intracytoplasmic injection of sperm (ICSI) and cryopreservation. Cryopreservation played an important role in infertility treatment as it has made ARTs more effective as well as more flexible. Cryopreservation of sperm and embryos can be performed efficiently, and both have been part of routine in vitro fertilization (IVF) procedures for a long time (David et al., 2012). However, cryopreservation of oocytes has not become a routine procedure until recently, even though the first successful pregnancies were pioneered more than two decades ago, in Australia and Europe, and more than a decade ago in the United States (Chen, 1986; Trounson and Mohr, 1983). The procedure offers benefit in a number of clinical, social, and logistic problems including women with premalignant/malignant conditions where disease or its treatment may have negative impact on future fertility (Del Mastro and Venturini, 2006; Rao et al., 2004). Women with conditions resulting in oophorectomy or irreversible ovarian failure (such as chemotherapy, radiotherapy, or certain genetic disorders) are also potential candidates for oocyte freezing. Another reason to freeze oocytes is to provide an option to delay childbirth. Additionally, oocyte freezing could be helpful in donor oocyte programs, which have several limitations in their efficiency due to several factors such as expense, availability of suitable donors, delay in synchronization between donor and recipient, and so on. So, a successful oocyte freezing program can help in establishment of oocyte banks, which would help couple as well as ART clinic by providing compatible oocytes from donor immediately, thus would eliminate the problem of long waiting list. Introduction of new vitrification technique has made the success rates for actual conception more reliable than the earlier method of slow freezing. With improved results of vitrification technique, and various studies reporting improved pregnancy rates, American Society of Reproductive Medicine has recently declared that oocyte cryopreservation should no longer be considered experimental for medical indications, outlying elective oocyte cryopreservation (Practice Committee of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology, 2013). Since then oocyte freezing and egg banking have been proposed for various new horizons of indications. In our current study, we aimed to evaluate outcomes of oocyte cryopreservation (using vitrification technique), observed in an oocyte donation program by comparing results in fresh and vitrified oocyte donor cycles. It would help to elucidate the potential of this technology in establishment of oocyte cryo-banking and would also provide an efficient treatment procedure with additional benefits to recipients as well as providing basis to apply it for different indications. MATERIAL AND METHODS A retrospective observational study was conducted at tertiary IVF center over the period of 1 year (from April 2016 to March 2017). Totaling 60 oocyte donation cycles were included in the study. Out of these 38 were fresh and 22 were vitrified oocytes cycles, respectively. A written consent was obtained from all the patients participating in the study. Ethical clearance was taken from institutional review board. Ovarian stimulation for donors All donors included in our oocyte donation program were between 21 and 35 years of age and screening was done according to Indian Council of Medical Research (ICMR) guidelines. Controlled ovarian hyperstimulation for donors was performed as follows: flexible antagonist protocol was used. Injection gonadotropins (highly purified hmg or recombinant FSH) were started from second day of menstrual cycle after a baseline scan. Dose of gonadotropins was decided by age, body mass index, antral follicle count, S.AMH, and previous response to stimulation. Monitoring of ovarian stimulation was done by serial ultrasonography performed at the interval of 2 3 days. GnRH antagonist (inj. Cetrorelix 0.25 mg S/C daily) was started when leading follicle reached >14 mm in diameter and continued till the day of trigger. Injection HCG 10,000 IU given intramuscularly to trigger final oocyte maturation when at least three follicles reached 17 mm in diameter. In case the number of follicles on the day of trigger was more than 14, injection triptorelin 0.2 mg subcutaneous was given in place of HCG in order to prevent risk of ovarian hyperstimulation syndrome (OHSS). Transvaginal ultrasoundguided oocyte retrieval was done after 34 hours of injection HCG or triptorelin administration under anesthesia. 10
Laboratory procedures The collected oocytes were washed and kept for 2 hours of postretrieval incubation. Denuding was done chemically (with hyaluronidase exposure for 30 seconds) and mechanically. Maturity of oocytes was confirmed. Oocytes containing polar body were considered as mature, that is, metaphase II (MII) oocytes and selected. In our study, only in vivo matured (MII) oocytes were used. All MII oocytes were allocated into two groups. Oocytes for cryopreservation were allocated to group vitrified oocytes and remaining were maintained in culture and assigned to the group fresh oocytes. Technique of cryopreservation Vitrification technique using Cryotop device was used for oocyte freezing. The first step of vitrification, that is, equilibration of oocytes was done in equilibration medium (basal medium with 7.5% (v/v) ethylene glycol and 7.5% (v/v) dimethyl sulfoxide, i.e., DMSO) at room temperature for 15 minutes. Then oocytes were transferred into the vitrification medium (basal medium with 15% (v/v) ethylene glycol, 15% (v/v) DMSO, and 0.5 M sucrose) at room temperature for 45 60 seconds. The basal medium used was HEPES-buffered medium supplemented with 20% (v/v) serum protein substitute. The cryoprotectants-treated oocytes were loaded with minimal volume onto a fine polypropylene strip (Cryotop; Kitazato BioPharma Co.). Finally, polypropylene strips carrying oocytes were then submerged into liquid nitrogen. For the warming process, the polypropylene strips with vitrified oocytes were removed from the liquid nitrogen and were immersed directly into 0.8 ml of thawing solution (20% HEPES-buffered medium with (v/v) serum protein substitute and 1.0 M sucrose) at 370 C for 1 minute. Oocytes were then picked up and transferred into 0.3 ml of the dilution solution (HEPES-buffered medium with 20% (v/v) SPS and 0.5 M sucrose) for 3 minutes at room temperature. Oocytes were subsequently washed in 0.3 ml washing solution (HEPES-buffered medium with 20% (v/v) SPS- 5 minutes in washing solution 1 (WS1) and 5 minutes in washing solution 2 (WS2) subsequently at room temperature. Solutions used for vitrification technique were obtained from Kitazato (Tokyo, Japan). Oocyte survival was assessed under inverted microscope and survived oocytes were kept in culture media at 37 C and 5% CO 2 for 2 3 hours before ICSI. Subsequently, oocytes were inseminated by ICSI using sperm from the corresponding recipient s husband. Then, oocytes were checked after about 16 17 hours postfertilization for pronuclei formation (first check) and those displaying presence of two pronuclei and two polar bodies were cultured further. Embryos were evaluated on day 3 morphologically and assessed for development and quality. Good quality day-3 embryos were defined as six to eight cells and <10% fragmentation and were selected for transfer. Preparation of recipients All oocytes retrieved from a single donor were donated to a single compatible recipient. Preparation of recipients was performed using a hormone replacement protocol. Injection leuprolide acetate depot (3.75 mg) intramuscular was given in the midluteal phase of proceeding cycle. Downregulation was confirmed by measuring endometrial thickness and Serum E2 level (ET <5 mm and S. E2 <50 pg/ml). Endometrial preparation was done with incremental doses of estradiol valerate starting from 2 mg/day to a maximum of 8 mg/day. Endometrial thickness was monitored by transvaginal ultrasound. Addition of vaginal micronized progesterone 200 mg capsule thrice a day was done with adequate endometrial preparation from the day of donor oocyte retrieval and ICSI in fresh cycle. In case of vitrified oocytes, warming of oocytes followed by ICSI insemination was done on day of starting progesterone. Pregnancy was confirmed with blood test (S. Beta HCG) after 14 days from embryo transfer. A clinical pregnancy was defined as presence of cardiac activity on ultrasound performed between 6 and 7 gestational weeks. STATISTICAL ANALYSIS All the statistical analysis was done using IBM SPSS version 16.0 (Armonk, NY, USA). Z-test for the significance of difference in mean was used to compare age between the two groups. Z-test for the significance of difference between the two proportions was used to compare the categorical/dichotomous variable among the groups (Yadav et al., 2019). The p value <0.05 was considered statistically significant. RESULTS A total of 600 oocytes were studied. Out of these 226 were vitrified oocytes and 374 were fresh oocytes. Both the groups were compared in terms of baseline characteristics and stimulation parameters. A total of 226 oocytes were vitrified and warmed for 22 recipients. After warming 218 oocytes survived (survival rate 96.4%). All survived oocytes were inseminated with ICSI and 188 oocytes were fertilized normally resulting in fertilization rate of 86.2%. A total of 176 embryos were formed on day three (embryo formation rate 93.6%). Two Day 3 good quality embryos were transferred for all the patients. Out of 22 recipients, 15 patients showed a positive beta HCG after 14 days of embryo transfer and 7 patients came negative. Out of the positive patients, 1 patient showed fall in beta HCG levels later on resulting in biochemical pregnancy; 14 patients showed presence of cardiac activity in ultrasonography at 6 7 weeks, suggesting a clinical pregnancy rate of 63.6%. Results of frozen-thawed oocyte donor cycles were compared with fresh donation cycles. (Tables 1 3) In case of fresh cycles, total of 374 oocyte cumulus complexes were obtained. Out of these 326 mature oocytes underwent insemination with ICSI. Table 1. Baseline characteristics and stimulation details of both the groups. Baseline characteristics Vitrified Fresh p-value Donor age 25.35 ± 5.3 24.5 ± 5.8 0.46 Mean age of recipients 38.5 ± 2.8 38.4 ± 3.6 0.76 Age of male partner 40.35 ± 1.86 40.82 ± 1.89 0.966 Stimulation details Length of stimulation 10.97 ± 1.7 10.35 ± 2.2 0.52 Total FSH (IU) 1208.8 ± 640.8 1275 ± 480.5 0.52 Total hmg (IU) 815.3 ± 219.4 782 ± 240.4 0.43 11
Table 2. Laboratory data of vitrified and fresh oocyte donation cycles. Vitrified cycles Fresh cycles p-value No. of oocytes 226 374 (COCs) No. of oocytes survived 218 NA Oocyte survival rate 96.4% NA No. of oocytes for ICSI 218 326 No. of fertilized oocytes 188 272 Fertilization Rate 86.2% 83.4% 0.38 Nonsignificant Embryo formation rate 93.6% (176/188) 92.6% (252/272) 0.69 Nonsignificant COCs: cumulus oocyte complexes. A total of 272 oocytes fertilized after ICSI, in comparison to 184 in vitrified oocyte group. So, the fertilization rate was 83.4% and 86.2% in fresh and vitrified group, respectively. Embryo formation rate on Day 3 was 92.6% (252 embryos formed out of 272), which was comparable to vitrified group. Similar to the vitrified group, two Day 3 good quality embryos were transferred for all patients. Out of 38 recipients, 23 showed a positive result with serum beta HCG and all patients reached up to sonography suggestive of presence of cardiac activity, with no recorded biochemical pregnancy. So, the clinical pregnancy rate was 60.5% in fresh group and 63.6% in vitrified group. DISCUSSION Oocyte donation is an important fertility treatment option for many couples, including women with advanced maternal age or women with decreased ovarian reserve due to variety of reasons. Fresh oocyte donation cycle is an established ART technique. However, several factors affect efficiency of successful oocyte donor program like availability of suitable donors, expense, delay in synchronization between donor and recipient, and inability to quarantine oocytes. With the help of successful oocyte freezing program some of these difficulties can be alleviated (Trokoudes et al., 2011). Though oocyte cryopreservation took a longer time to become a routine ART procedure, in comparison of embryo and sperm cryopreservation, many reports have shown successful pregnancies following oocyte freezing, especially with the new vitrification technique of oocyte cryopreservation (Smith et al., 2010). Our study compared the outcomes of vitrified and fresh oocytes in donor cycles. Both clinical and laboratory results obtained in the study suggests that oocyte cryopreservation can be performed with reproducible success, thus vitrification technique provides a potential option for establishment of an Oocyte-bank. However, the most important prerequisite for a successful banking program is to have an efficient freezing warming technique. The recent vitrification technique is a nonequilibrium cooling, which utilizes very high concentrations of cryoprotectants that Table 3. Clinical data of vitrified and fresh oocyte donation cycles. Vitrified oocytes Fresh oocytes p-value No. of donors 22 38 No. recipients 22 38 Biochemical pregnancy 6.2% (1/16) 0 Clinical pregnancy rate 63.6% 60.5% p Value = 0.81 Nonsignificant solidify without forming ice crystals, thus avoids the problems as seen with the slow freezing technique used previously. Most challenging step in cryopreservation probably is to maintain plasma and membrane integrity by preventing ice crystal formation. Vitrification has overcome two main limiting factors for achieving an adequate cryopreservation: ice crystal formation and chilling injury (Vajta and Kuwayama, 2006). To prevent ice crystal formation, various permeating and non-permeating cryoprotectants are used at higher concentrations. However, some of these cryoprotectants themselves are toxic at higher concentrations. So, to avoid these problems, an intrinsic balance is needed between the concentrations and exposure time of cryoprotectants. On the other hand, vitrification minimizes chilling injury by using high cooling rate (Vajta and Nagy, 2006). Vitrification has gained popularity in freezing the oocytes due to its superiority to the slow-freezing method, in form of better oocyte survival rate, fertilization, and embryonic development in vitro. In our study, survival rate of oocytes after warming was 96.4%. Employing vitrification several authors have also reported a much improved survival rate. A study by Cobo et al. (2008) also compared the outcomes achieved with fresh and cryopreserved donor oocytes using Cryotop device and noted a survival rate of 96.7%. When comparing the fertilization rate, in case of vitrified oocytes it was 86.2% while 83.4% fresh oocytes were fertilized normally. So, although a slightly higher fertilization rate was noted in case of vitrified oocytes, there was no statistically significant difference (p value = 0.38). Similar to our results, study by Nagy et al. (2009) also reported that fertilization rates were similarly high in both groups, although a trend may be noted for higher fertilization in the frozen/warmed oocyte group. Embryo formation rate in our study was found to be 93.6% in case of vitrified oocytes and 92.6% for fresh oocytes with insignificant difference statistically (p value = 0.69). Similar to our results, study by Rienzi et al. also showed that oocyte vitrification procedure followed by ICSI is not inferior to fresh insemination procedure, with regard 12
to embryo developmental rates (Rienzi et al., 2010). Finally, the main outcome studied was implantation potential of embryos derived from vitrified and fresh oocytes resulting in viable pregnancies. In our study, the clinical pregnancy rate in vitrified group was 63.6% and in fresh oocyte group was 60.5%. However, the difference was found to be statistically insignificant (p value = 0.81). Consistent with the results of our study, several studies have also demonstrated a reproducible success with oocyte cryopreservation. In a recent meta-analysis by Potdar et al. in 2014 on oocyte vitrification and post warming fertility outcomes, while comparing vitrified with fresh oocytes, no statistically significant difference was observed in fertilization, cleavage, and clinical pregnancy rates, but ongoing pregnancy rate was reduced in the vitrified group (odds ratio 0.74). However, marked clinical and statistical heterogeneity was noted in these studies. The review highlighted the scientific excellence that has been achieved over the decades. It suggests that cryopreservation is effective and safe, but individual units need to determine their safety and efficacy using vitrification techniques. Similarly, in a retrospective study, Trokoudes et al. (2011) have compared fertilization and cleavage rates, embryo quality and clinical results obtained from vitrified compared with fresh oocytes. They concluded that oocyte donation using vitrified oocytes can provide the same quality of embryos, pregnancy, and implantation potential as fresh oocyte donations (Trokoudes et al., 2011). Despite the excellent results with cryopreservation, several concerns have been raised. One of the concerns was expressed that cooling or freezing may affect meiotic spindle integrity as well as may cause other cellular and subcellular alterations resulting in chromosomal abnormalities. However, studies are reassuring in this regard. In a study Noyes et al. (2009), constructed a database to verify live-born infants born after oocyte freezing and 58 reports (1986 2008) were reviewed, which included 609 live-born babies. Additionally, 327 other live births were verified. Of the total 936 live borns, 1.3% (12) was noted to have birth anomalies. No difference was noted in congenital anomalies compared with naturally conceived infants. They concluded with more live-born data accumulating, this procedure may become mainstream as a fertility preservation option, particularly for women diagnosed with malignancy requiring cytotoxic therapy. Finally, observations in our study, conclude that all the clinical and laboratory outcomes for embryos obtained from vitrified oocytes such as oocyte fertilization rate, embryo formation rate, and clinical pregnancy were similar to results achieved with fresh oocytes indicating that, developmental potential of embryos derived from vitrified oocytes is conserved. So, cryopreservation can be provided as a useful tool for achieving highly successful outcomes in an oocyte donor program. Cobo A, Kuwayama M, Pérez S, Ruiz A, Pellicer A, Remohí J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril. 2008 June;89(6):1657 64. Epub 2007 September 24. David KG, Ariel W, Colin MH, Zeev S. Textbook of Assisted Reproductive Techniques. 4th ed. New York: Parthenon Publishing Group; 2012. Del Mastro L, Venturini M. Fertility preservation strategies for breast cancer patients. J Clin Oncol. 2006;24:4220 1. Nagy ZP, Chang CC, Shapiro DB, Bernal DP, Elsner CW, Mitchell-Leef D. Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking. Fertil Steril. 2009 August;92(2):520 6. Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed Online. 2009 June;18(6):769 7. Potdar N, Gelbaya TA, Nardo LG. Oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis. Reprod Biomed Online. 2014;29:159 76. Practice Committee of American Society for Reproductive Medicine; Society for Assisted Reproductive Technology. Mature oocyte cryopreservation: a guideline. Fertil Steril. 2013;99:37 43. Rao GD, Chian RC, Son WS, Gilbert L, Tan SL. Fertility preservation in women undergoing cancer treatment. Lancet. 2004;363: 1829 30. Rienzi L, Romano S, Albricci L, et al. Embryo development of fresh versus vitrified metaphase II oocytes after ICSI: a prospective randomized sibling-oocyte study. Hum Reprod (Oxford, England). 2010;25(1):66 73. Smith GD, Serafini PC, Fioravanti J, et al. Prospective randomized comparison of human oocyte cryopreservation with slow-rate freezing or vitrification. Fertil Steril. 2010;94:2088 95. Trokoudes KM, Pavlides C, Zhang X. Comparison outcome of fresh and vitrified donor oocytes in an egg-sharing donation program. Fertil Steril. 2011 May;95(6):1996 2000. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305: 707 9. Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology. 2006;65:236 44. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online. 2006;12:779 9. Yadav SK, Singh S, Gupta R. Test for inference: categorical data I. In: Biomedical Statistics. Singapore: Springer; 2019. REFERENCES Chen C. Pregnancy after human oocyte cryopreservation. Lancet. 1986;1:884 6. 13